Type IV pili are extracellular bacterial filaments required for attachment to surfaces and for pathogenesis in many plant, animal, and human infections (including bacterial meningitis caused by Neisseria meningitidis, gonorrhea caused by N. gonorrhoeae, and lung infections of cystic fibrosis patients caused by Pseudomonas aeruginosa). Through the work of the intracellular ATPase PilT, pili are retracted into the cell from which they emerge. Retraction leads to motility on surfaces and activates signaling pathways in eukaryotic target cells. Based on structural and mutagenesis data we suggest that ATP binding to PilT induces large-scale movements of individual domains, which adjusts the interface between subunits and promotes synergistic motor movements. Because unchecked retraction leads to non-piliated cells, the force of retraction is counterbalanced by other proteins with similar N-terminal sequences to Type IV prepilins. The three specific aims of this proposal are designed to improve our understanding of the mechanism of pilus retraction and counterbalance. We will take a simultaneous bottom-up &top-down approach that capitalizes on our expertise in protein structure/function analysis. Specifically, we will (1) carry out X-ray crystallography and small angle scattering studies of P. aeruginosa and N. gonorrhoeae PilT bound to various nucleotide analogues plus structure-based mutagenesis and phenotypic analysis of the resulting strains (2) identify the minimal set of pilus biogenesis proteins by expression in a non-piliated organism and (3) carry out protein crystallography and site directed mutagenesis of the low abundance prepilin-like proteins. The overall result of these experiments will be a better understanding of the molecular mechanism of retraction of the widespread bacterial virulence factor Type IV pili. Ultimately this knowledge will be used in designing approaches to control or block this pathway, thereby preventing the spread of many microbial diseases.